1 essenbioscience.com/IncuCyte IncuCyte ™ Reporter Gene Assay Real-time measurement of reporter gene expression Real-time visualization and analysis enable interrogation of cell morphology changes with signalling pathway activation. Measure signaling pathway activation and activity of any promoter driven recombinant GFP/RFP reporter gene expression in real time. Save cost and time – no terminal reaction substrate and defining end points a priori. Schematic representation of the NFκB reporter gene concept. Cells are transfected or infected with a vector containing the NF-κB transcriptional response element just upstream of the GFP reporter gene. Cells are then treated with TNFα which activates the TNFα receptor followed by degradation of IκBα, and translocation of the NFκB transcription factor subunits to the nucleus where they participate in driving the expression of the GFP transgene. Green fluorescing cells can be kinetically imaged using the IncuCyte ® ZOOM Instrument and the number of fluorescing cells can be quantified. HEK293 cells were transiently transfected with an NFkB-GFP reporter gene construct, serum starved, and treated with the indicated amounts of TNFα. Images were acquired every 2 hours. Summed intensity is reported as a marker for NFkB promoter activity.